PROJECT SUMMARY MicroRNAs (miRNAs) have emerged as powerful regulators of the genome and, through concerted efforts to identify their function and evaluate their ability to alter cell growth in vitro and in vivo, some have gained favor as potential therapeutics. Although these miRNA-based approaches can revolutionize the way that tumors are diagnosed and treated, the challenges that remain with regard to in vivo delivery of miRNAs must be tackled. To address these challenges, we have pioneered a robust and efficient method for ligand-mediated delivery of miRNAs (FolamiRs), which has removed the toxic delivery vehicle. In this application we propose to enhance and characterize FolamiR targeting through facilitating intracellular endosomal-mediated release using the small molecule ionophore nigericin conjugated onto the FolamiRs, and through characterizing an intercellular bystander effect between folate receptor positive and folate receptor negative cells. To achieve these goals, the following three Specific Aims are proposed: I. Facilitate endosomal release of FolamiRs through synthesis of alternative Nig-FolamiR chemistries, II. Evaluate in vivo efficacy and safety of Nig-FolamiRs, and III. Evaluate the mechanism involved in the observed bystander effect following FolamiR treatment. Our extensive preliminary evidence supports all three Aims. We show that the clinically relevant miRNA, miRNA-34a (miR-34a) is selectively targeted to the tumor, enters the tumorigenic cells, downregulates target genes, and prevents growth of tumors in vivo. Success is dependent on rapid tumor uptake that is mediated by tagging miR-34a to a relevant ligand, in this case folate (FolamiR-34a). Indeed, the folate receptor is specifically upregulated on various tumors including breast, lung, ovarian, kidney, and colon cancers, which can facilitate tumor specific uptake of FolamiRs. Moreover, using a small molecule ionophore, nigericin, we show that intracellular concentrations of FolamiRs can be enhanced due to endosomal release. The clinical utility and safety profile of nigericin will be evaluated in vivo. Additional chemistries of nigericin-FolamiR conjugates will be synthesized and evaluated, including inclusion of multiple nigericin molecules per FolamiR to enhance endosomal swelling and rupture, and chemistries to prevent nonspecific release of nigericin from FolamiRs in circulation. Finally, the mechanism involved in the intercellular bystander effect will be determined. Overall, this innovative delivery method has implications well beyond miRNA delivery. One could imagine attaching any small RNA onto folate and achieving fast, and tumor-specific uptake. Similarly, the small molecule ionophore being tested for endosomal mediated escape has the potential to positively impact many therapeutics that are sequestered into the endosomes. Again, highlighting the broad, and powerful impact that these innovative studies will have.